The important process of communication between astrocytes and neurons is exocytotic release of gliotransmitters (such as glutamate, neuroactive peptides and ATP) from membrane-bound vesicles into the extracellular space. To explore whether stimuli that increase [Ca²⁺]_i trigger vesicular ATP release from astrocytes, we used HEK-293T cells transfected with P₂X₃ receptor, used as sniffers for ATP release from astrocytes. Glutamate stimulation of astrocytes was followed by an increase in the incidence of small transient inward currents in sniffers, reminiscent of postsynaptic quantal events observed at synapses. Their incidence was highly dependent on extracellular Ca²⁺. This indicates that glutamate-stimulated ATP release from astrocytes was most likely exocytotic. Prior fusing with the plasma membrane in the process of exocytosis, membrane-bound vesicles are transported through the cytoplasm. Their trafficking and consequently release of their content may be changed in altered physiological conditions, therefore affecting the physiological status of neurons. We studied the prefusion mobility of fluorescently labeled peptidergic ANP vesicles (atrial natriuretic peptide; ANP) in the cytoplasm of single rat and mouse astrocytes in culture. We found out that delivery of vesicles to the plasma membrane for exocytosis involves an interaction with the cytoskeleton, in particular microtubules and actin filaments, which is similar to neurons and excitable secretory cells. Some of the membrane-bound vesicles are retrieved from the plasma membrane to be recycled back into the cytosol. Postfusion transport of glutamatergic and peptidergic vesicles was studied by labeling vesicles with antibodies against specific membrane or luminal vesicle proteins. Trafficking velocity of prefusion and postfusion (recycling) vesicles differ in velocity for one order of magnitude and the traffic of diverse vesicles appears to be differently regulated by calcium ions.